Familial scaphocephaly syndrome caused by a novel mutation in the FGFR2 tyrosine kinase domain.
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Abnormalities, Multiple↗
Biomedical subjects
J F Bateman
Publications and source records attributed to J F Bateman.
A new FACIT of the collagen family: COL21A1.
Interrogation of the Human Genome data for sequences related to the von Willebrand factor A-domain module identified a previously unreported 4.1 kb full-length cDNA that is predicted to encode a new member of the collagen superfamily of extracellular matrix proteins, collagen XXI. The domain organization of collagen XXI comprised an N-terminal signal sequence, followed by single von Willebrand factor A-domain and thrombospondin domains, and an interrupted collagen triple helix. The organization of these motifs predict that collagen XXI is a new member of the FACIT collagen sub-family. Expression analysis indicated that COL21A1 mRNA is present in many tissues including heart, stomach, kidney, skeletal muscle and placenta, and radiation hybrid mapping localized the COL21A1 gene to 6p11-12.
Amino Acid Motifs↗
The N-terminal N5 subdomain of the alpha 3(VI) chain is important for collagen VI microfibril formation.
Collagen VI assembly is unique within the collagen superfamily in that the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains associate intracellularly to form triple helical monomers, and then dimers and tetramers, which are secreted from the cell. Secreted tetramers associate end-to-end to form the distinctive extracellular microfibrils that are found in virtually all connective tissues. Although the precise protein interactions involved in this process are unknown, the N-terminal globular regions, which are composed of multiple copies of von Willebrand factor type A-like domains, are likely to play a critical role in microfibril formation, because they are exposed at both ends of the tetramers. To explore the role of these subdomains in collagen VI intracellular and extracellular assembly, alpha 3(VI) cDNA expression constructs with sequential N-terminal deletions were stably transfected into SaOS-2 cells, producing cell lines that express alpha 3(VI) chains with N-terminal globular domains containing modules N9-N1, N6-N1, N5-N1, N4-N1, N3-N1, or N1, as well as the complete triple helix and C-terminal globular domain (C1-C5). All of these transfected alpha 3(VI) chains were able to associate with endogenous alpha 1(VI) and alpha 2(VI) to form collagen VI monomers, dimers, and tetramers, which were secreted. Importantly, cells that expressed alpha 3(VI) chains containing the N5 subdomain, alpha 3(VI) N9-C5, N6-C5, and N5-C5, formed microfibrils and deposited a collagen VI matrix. In contrast, cells that expressed the shorter alpha 3(VI) chains, N4-C5, N3-C5, and N1-C5, were severely compromised in their ability to form end-to-end tetramer assemblies and failed to deposit a collagen VI matrix. These data demonstrate that the alpha 3(VI) N5 module is critical for microfibril formation, thus identifying a functional role for a specific type A subdomain in collagen VI assembly.
Collagen↗
A dominant interference collagen X mutation disrupts hypertrophic chondrocyte pericellular matrix and glycosaminoglycan and proteoglycan distribution in transgenic mice.
Collagen X transgenic (Tg) mice displayed skeleto-hematopoietic defects in tissues derived by endochondral skeletogenesis.(1) Here we demonstrate that co-expression of the transgene product containing truncated chicken collagen X with full-length mouse collagen X in a cell-free translation system yielded chicken-mouse hybrid trimers and truncated chicken homotrimers; this indicated that the mutant could assemble with endogenous collagen X and thus had potential for dominant interference. Moreover, species-specific collagen X antibodies co-localized the transgene product with endogenous collagen X to hypertrophic cartilage in growth plates and ossification centers; proliferative chondrocytes also stained diffusely. Electron microscopy revealed a disrupted hexagonal lattice network in the hypertrophic chondrocyte pericellular matrix in Tg growth plates, as well as altered mineral deposition. Ruthenium hexamine trichloride-positive aggregates, likely glycosaminoglycans (GAGs)/proteoglycans (PGs), were also dispersed throughout the chondro-osseous junction. These defects likely resulted from transgene co-localization and dominant interference with endogenous collagen X. Moreover, altered GAG/PG distribution in growth plates of both collagen X Tg and null mice was confirmed by a paucity of staining for hyaluronan and heparan sulfate PG. A provocative hypothesis links the disruption of the collagen X pericellular network and GAG/PG decompartmentalization to the potential locus for hematopoietic failure in the collagen X mice.
Animals↗
Gelatinase A (MMP-2) activation by skin fibroblasts: dependence on MT1-MMP expression and fibrillar collagen form.
The respective requirements of collagen and MT1-MMP in the activation of MMP-2 by primary fibroblast cultures were explored further. Three-dimensional gels enriched in human collagen types I and III or composed of recombinant human type II or III collagen, caused increased MT1-MMP production (mRNA and protein) and induced MMP-2 activation. Only marginal induction was seen with dried monomeric collagen confirming the need for collagen fibrillar organisation for activation. To our surprise, relatively low amounts (as low as 25 microg/ml) of acid soluble type I collagen added to fibroblast cultures also induced potent MMP-2 activation. However, the requirement for collagen fibril formation by the added collagen was indicated by the inhibition seen when the collagen was pre-incubated with a fibril-blocking peptide, and the reduced activation seen with alkali-treated collagen preparations known to have impaired fibrilisation. Pre-treatment of the collagen with sodium periodate also abrogated MMP-2 activation induction. Further evidence of the requirement for collagen fibril formation was provided by the lack of activation when type IV collagen, which does not form collagen fibrils, was added in the cultures. Fibroblasts derived from MT1-MMP-deficient mice were unable to activate MMP-2 in response to either three-dimensional collagen gel or added collagen solutions, compared to their littermate controls. Collectively, these data indicate that the fibrillar structure of collagen and MT1-MMP are essential for the MMP-2 activational response in fibroblasts.
Animals↗
The molecular genetics of inherited cartilage disease.
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Cartilage Diseases↗
UNCL, the mammalian homologue of UNC-50, is an inner nuclear membrane RNA-binding protein.
We isolated a mammalian homologue of the C. elegans gene unc-50 that we have named UNCL. The 777 kb rat UNCL cDNA encodes a 259 amino acid protein that is expressed in a wide variety of tissues with highest mRNA levels in brain, kidney and testis. Hydropathy plot analysis and in vitro translation experiments with microsomal membranes indicate that UNCL is a transmembrane protein. Hemagglutinin tagged UNCL was stably transfected into SaOS-2 osteosarcoma cells and exhibited a nuclear rim staining pattern which was retained following extraction with 1% Triton X-100, suggesting that UNCL localizes to the inner nuclear membrane. UNCL-HA was extractable in 350 mM NaCl, suggesting that UNCL is not associated with the nuclear matrix. Homopolymer RNA-binding assays performed on in vitro translated UNCL protein and 'structural modeling by homology' suggest that UNCL binds RNA via an amino-terminal RNA Recognition-like Motif. Since unc-50 is required for expression of assembled muscle-type nicotinic receptors in the nematode we investigated whether UNCL had a similar function for mammalian nicotinic receptors. When UNCL was co-expressed with neural nicotinic receptors in Xenopus oocytes or COS cells it increased expression of functional cell surface receptors up to 1. 6-fold. We conclude that UNCL is a novel inner nuclear membrane protein that associates with RNA and is involved in the cell-surface expression of neuronal nicotinic receptors. UNCL plays a broader role because UNCL homologues are present in two yeast and a plant species, none of which express nicotinic receptors and it is also found in tissues that lack nicotinic receptors.
Amino Acid Sequence↗
Molecular diagnosis of Stickler syndrome: a COL2A1 stop codon mutation screening strategy that is not compromised by mutant mRNA instability.
We have developed a novel strategy for screening families with type 1 Stickler syndrome due to COL2A1 nonsense mutations, using a modified RNA-based protein truncation test. To overcome the problem of the unavailability of collagen II-producing cartilage cells, reverse transcription polymerase chain reaction (RT-PCR) was performed on the illegitimate transcripts of accessible cells (lymphoblasts and fibroblasts), which were pre-incubated with cycloheximide to prevent nonsense-mutation-induced mRNA decay. The five overlapping RT-PCR fragments covering the COL2A1 coding region were then transcribed and translated in vitro to identify smaller truncated protein products which result from a premature stop codon. This method was used to screen a 4-generation Stickler family and a protein truncating mutation was identified, which was present in all affected individuals. Targeted sequencing identified the mutation as a G(+1) to A substitution at the 5' splice donor site of intron 25, which led to the activation of a cryptic splice site 8-bp upstream causing aberrant mRNA splicing and a translational frameshift that introduced a premature stop codon. Mutant mRNA was undetectable without cycloheximide protection, demonstrating that the mutant mRNA was subjected to nonsense-mediated mRNA decay. As well as providing further evidence that type 1 Stickler syndrome results from COL2A1 premature stop codon mutations, this study suggests mutant mRNA instability leading to haploinsufficiency may also be an important, but previously unrecognized, molecular basis of Stickler syndrome. This rapid new test for COL2A1 nonsense mutations is of particular clinical importance to Stickler syndrome families, where the identification of individuals who are at risk of this potentially preventable form of blindness will allow them to undergo regular ophthalmological surveillance and preventative or early ameliorative treatment.
Abnormalities, Multiple↗
Mammalian skeletogenesis and extracellular matrix: what can we learn from knockout mice?
Formation of the vertebrate skeleton and the proper functions of bony and cartilaginous elements are determined by extracellular, cell surface and intracellular molecules. Genetic and biochemical analyses of human heritable skeletal disorders as well as the generation of knockout mice provide useful tools to identify the key players of mammalian skeletogenesis. This review summarises our recent work with transgenic animals carrying ablated genes for cartilage extracellular matrix proteins. Some of these mice exhibit a lethal phenotype associated with severe skeletal defects (type II collagen-null, perlecan-null), whereas others show mild (type IX collagen-null) or no skeletal abnormalities (matrilin-1-null, fibromodulin-null, tenascin-C-null). The appropriate human genetic disorders are discussed and contrasted with the knockout mice phenotypes.
Animals↗
Proteasomal degradation of unassembled mutant type I collagen pro-alpha1(I) chains.
We have previously shown that type I procollagen pro-alpha1(I) chains from an osteogenesis imperfecta patient (OI26) with a frameshift mutation resulting in a truncated C-propeptide, have impaired assembly, and are degraded by an endoplasmic reticulum-associated pathway (Lamandé, S. R., Chessler, S. D., Golub, S. B., Byers, P. H., Chan, D., Cole, W. G., Sillence, D. O. and Bateman, J. F. (1995) J. Biol. Chem. 270, 8642-8649). To further explore the degradation of procollagen chains with mutant C-propeptides, mouse Mov13 cells, which produce no endogenous pro-alpha1(I), were stably transfected with a pro-alpha1(I) expression construct containing a frameshift mutation that predicts the synthesis of a protein 85 residues longer than normal. Despite high levels of mutant mRNA in transfected Mov13 cells, only minute amounts of mutant pro-alpha1(I) could be detected indicating that the majority of the mutant pro-alpha1(I) chains synthesized are targeted for rapid intracellular degradation. Degradation was not prevented by brefeldin A, monensin, or NH(4)Cl, agents that interfere with intracellular transport or lysosomal function. However, mutant pro-alpha1(I) chains in both transfected Mov13 cells and OI26 cells were protected from proteolysis by specific proteasome inhibitors. Together these data demonstrate for the first time that procollagen chains containing C-propeptide mutations that impair assembly are degraded by the cytoplasmic proteasome complex, and that the previously identified endoplasmic reticulum-associated degradation of mutant pro-alpha1(I) in OI26 is mediated by proteasomes.
Amino Acid Sequence↗
Bethlem myopathy and engineered collagen VI triple helical deletions prevent intracellular multimer assembly and protein secretion.
Mutations in the genes that code for collagen VI subunits, COL6A1, COL6A2, and COL6A3, are the cause of the autosomal dominant disorder, Bethlem myopathy. Although three different collagen VI structural mutations have previously been reported, the effect of these mutations on collagen VI assembly, structure, and function is currently unknown. We have characterized a new Bethlem myopathy mutation that results in skipping of COL6A1 exon 14 during pre-mRNA splicing and the deletion of 18 amino acids from the triple helical domain of the alpha1(VI) chain. Sequencing of genomic DNA identified a G to A transition in the +1 position of the splice donor site of intron 14 in one allele. The mutant alpha1(VI) chains associated intracellularly with alpha2(VI) and alpha3(VI) to form disulfide-bonded monomers, but further assembly into dimers and tetramers was prevented, and molecules containing the mutant chain were not secreted. This triple helical deletion thus resulted in production of half the normal amount of collagen VI. To further explore the biosynthetic consequences of collagen VI triple helical deletions, an alpha3(VI) cDNA expression construct containing a 202-amino acid deletion within the triple helix was produced and stably expressed in SaOS-2 cells. The transfected mutant alpha3(VI) chains associated with endogenous alpha1(VI) and alpha2(VI) to form collagen VI monomers, but dimers and tetramers did not form and the mutant-containing molecules were not secreted. Thus, deletions within the triple helical region of both the alpha1(VI) and alpha3(VI) chains can prevent intracellular dimer and tetramer assembly and secretion. These results provide the first evidence of the biosynthetic consequences of structural collagen VI mutations and suggest that functional protein haploinsufficiency may be a common pathogenic mechanism in Bethlem myopathy.
Adult↗
Interaction of collagen alpha1(X) containing engineered NC1 mutations with normal alpha1(X) in vitro. Implications for the molecular basis of schmid metaphyseal chondrodysplasia.
Collagen X is a short-chain homotrimeric collagen expressed in the hypertrophic zone of calcifying cartilage. The clustering of mutations in the carboxyl-terminal nonhelical NC1 domain in Schmid metaphyseal chondrodysplasia (SMCD) suggests a critical role for NC1 in collagen X structure and function. In vitro collagen X DNA expression, using T7-driven coupled transcription and translation, demonstrated that although alpha1(X) containing normal NC1 domains can form electrophoretically stable trimers, engineered SMCD NC1 missense or premature termination mutations prevented the formation of electrophoretically stable homotrimers or heterotrimers when co-expressed with normal alpha1(X). To allow the detection of more subtle interactions that may interfere with assembly but not produce SDS-stable final products, we have developed a competition-based in vitro co-expression and assembly approach. Our studies show that alpha1(X) chains containing SMCD mutations reduce the efficiency of normal alpha1(X) trimer assembly, indicating that interactions do occur between mutant and normal NC1 domains, which can impact on the formation of normal trimers. This finding has important implications for the molecular pathology of collagen X mutations in SMCD. Although we have previously demonstrated haploinsufficiency as one in vivo mechanism (Chan, D., Weng, Y. M., Hocking, A. M., Golub, S., McQuillan, D. J., and Bateman, J. F. (1998) J. Clin. Invest. 101, 1490-1499), the current study suggests dominant interference is also possible if the mutant protein is expressed in vivo. Furthermore, we establish that a conserved 13-amino acid aromatic motif (amino acids 589-601) is critical for the interaction between the NC1 domains, suggesting that this region may initiate assembly and the other NC1 mutations interfered with secondary interactions important in folding or in stabilizing the assembly process.
Base Sequence↗
Reliable and sensitive detection of premature termination mutations using a protein truncation test designed to overcome problems of nonsense-mediated mRNA instability.
The protein truncation test (PTT) is a mutation-detection method used to scan for premature termination (nonsense) mutations. PCR amplification of the DNA or mRNA source material is performed using forward primers containing a T7-promoter sequence and translation initiation signals such that the resultant products can be transcribed and translated in vitro to identify the smaller truncated protein products. mRNA is commonly used as the source material, but success of the PTT and other RNA-based mutation detection methods can be severely compromised by nonsense mutation-induced mRNA decay, a well-documented process that is often overlooked in mutation detection strategies. In this study, we develop an RNA-based PTT that overcomes the problem of mRNA decay by preincubating cells with cycloheximide to stabilise the mutant mRNA. The effectiveness of this method for mutation detection in abundant mRNAs was demonstrated in osteogenesis imperfecta fibroblasts by the protection of type I collagen (COL1A1) mRNA containing nonsense mutations that normally resulted in mutant mRNA degradation. Stabilisation of mutant mismatch repair gene (MLH1) mRNA was also observed in transformed lymphocytes from patients with hereditary nonpolyposis colorectal cancer (HNPCC). Importantly, our strategy also stabilised very low-level (or illegitimate) nonsense-containing transcripts in lymphoblasts from patients with Bethlem myopathy (COL6A1), familial adenomatous polyposis (APC), and breast cancer (BRCA1). The greatly increased sensitivity and reliability of this RT-PCR/PTT protocol has broad applicability to the many genetic diseases in which only blood-derived cells may be readily available for analysis.
Adaptor Proteins, Signal Transducing↗
Procollagen folding and assembly: the role of endoplasmic reticulum enzymes and molecular chaperones.
Procollagen assembly occurs within the endoplasmic reticulum, where the C-propeptide domains of three polypeptide alpha-chains fold individually, and then interact and trimerise to initiate folding of the triple helical region. This highly complex folding and assembly pathway requires the co-ordinated action of a large number of endoplasmic reticulum-resident enzymes and molecular chaperones. Disease-causing mutations in the procollagens disturb folding and assembly and lead to prolonged interactions with molecular chaperones, retention in the endoplasmic reticulum, and intracellular degradation. This review focuses predominantly on prolyl 1-hydroxylase, an essential collagen modifying enzyme, and HSP47, a collagen-specific binding protein, and their proposed roles as molecular chaperones involved in fibrillar procollagen folding and assembly, quality control, and secretion.
Collagen↗
Normal skeletal development of mice lacking matrilin 1: redundant function of matrilins in cartilage?
Matrilin 1, or cartilage matrix protein, is a member of a novel family of extracellular matrix proteins. To date, four members of the family have been identified, but their biological role is unknown. Matrilin 1 and matrilin 3 are expressed in cartilage, while matrilin 2 and matrilin 4 are present in many tissues. Here we describe the generation and analysis of mice carrying a null mutation in the Crtm gene encoding matrilin 1. Anatomical and histological studies demonstrated normal development of homozygous mutant mice. Northern blot and biochemical analyses show no compensatory up-regulation of matrilin 2 or 3 in the cartilage of knockout mice. Although matrilin 1 interacts with the collagen II and aggrecan networks of cartilage, suggesting that it may play a role in cartilage tissue organization, studies of collagen extractability indicated that collagen fibril maturation and covalent cross-linking were unaffected by the absence of matrilin 1. Ultrastructural analysis did not reveal any abnormalities of matrix organization. These data suggest that matrilin 1 is not critically required for cartilage structure and function and that matrilin 1 and matrilin 3 may have functionally redundant roles.
Animals↗
Aortic dissection, patent ductus arteriosus, iris hypoplasia and brachytelephalangy in a male adolescent.
We describe a 14-year-old male with dissection of the descending aorta, bilateral iris hypoplasia, striae distensae and brachytelephalangy, the latter being most marked in the thumbs. Inguinal herniae and a patent ductus arteriosus were surgically repaired in infancy. The pattern of abnormalities may constitute a previously undescribed syndrome. The proband died suddenly at the age of 17 years.
Abnormalities, Multiple↗
Collagen II is essential for the removal of the notochord and the formation of intervertebral discs.
Collagen II is a fibril-forming collagen that is mainly expressed in cartilage. Collagen II-deficient mice produce structurally abnormal cartilage that lacks growth plates in long bones, and as a result these mice develop a skeleton without endochondral bone formation. Here, we report that Col2a1-null mice are unable to dismantle the notochord. This defect is associated with the inability to develop intervertebral discs (IVDs). During normal embryogenesis, the nucleus pulposus of future IVDs forms from regional expansion of the notochord, which is simultaneously dismantled in the region of the developing vertebral bodies. However, in Col2a1-null mice, the notochord is not removed in the vertebral bodies and persists as a rod-like structure until birth. It has been suggested that this regional notochordal degeneration results from changes in cell death and proliferation. Our experiments with wild-type mice showed that differential proliferation and apoptosis play no role in notochordal reorganization. An alternative hypothesis is that the cartilage matrix exerts mechanical forces that induce notochord removal. Several of our findings support this hypothesis. Immunohistological analyses, in situ hybridization, and biochemical analyses demonstrate that collagens I and III are ectopically expressed in Col2a1-null cartilage. Assembly of the abnormal collagens into a mature insoluble matrix is retarded and collagen fibrils are sparse, disorganized, and irregular. We propose that this disorganized abnormal cartilage collagen matrix is structurally weakened and is unable to constrain proteoglycan-induced osmotic swelling pressure. The accumulation of fluid leads to tissue enlargement and a reduction in the internal swelling pressure. These changes may be responsible for the abnormal notochord removal in Col2a1-null mice. Our studies also show that chondrocytes do not need a collagen II environment to express cartilage-specific matrix components and to hypertrophy. Furthermore, biochemical analysis of collagen XI in mutant cartilage showed that alpha1(XI) and alpha2 (XI) chains form unstable collagen XI molecules, demonstrating that the alpha3(XI) chain, which is an alternative, posttranslationally modified form of the Col2a1 gene, is essential for assembly and stability of triple helical collagen XI.
Animals↗
A nonsense mutation in the carboxyl-terminal domain of type X collagen causes haploinsufficiency in schmid metaphyseal chondrodysplasia.
Type X collagen is a short-chain homotrimeric collagen expressed in the hypertrophic zone of calcifying cartilage. The clustering of mutations in the carboxyl-terminal NC1 domain in Schmid metaphyseal chondrodysplasia (SMCD) suggested a critical role for this type X collagen domain, but since no direct analysis of cartilage has been conducted in SMCD patients, the mechanisms of type X collagen dysfunction remain controversial. To resolve this problem, we obtained SMCD growth plate cartilage, determined the type X collagen mutation, and analyzed the expression of mutant and normal type X collagen mRNA and protein. The mutation was a single nucleotide substitution that changed the Tyr632 codon (TAC) to a stop codon (TAA). However, analysis of the expression of the normal and mutant allele transcripts in growth plate cartilage by reverse transcription PCR, restriction enzyme mapping, and a single nucleotide primer extension assay, demonstrated that only normal mRNA was present. The lack of mutant mRNA is most likely the result of nonsense-mediated mRNA decay, a common fate for transcripts carrying premature termination mutations. Furthermore, no mutant protein was detected by immunoblotting cartilage extracts. Our data indicates that a functionally null allele leading to type X collagen haploinsufficiency is the molecular basis of SMCD in this patient.
Animals↗